Technical Abstract:
Sugarcane geneticists routinely exchange and move a large number of clones by vegetative propagation across locations. They rely on morphological keys developed by the originating location to distinguish these clones. Since environments can influence these traits, genetically distinct clones, in particular those from the same geographical location may appear similar, thus causing some variety identity concern. DNA fingerprints or molecular identities are unique to any given clone and are not influenced by environments. Since 2005, we have genotyped 655 clones with 21 SSR markers using a high-throughput (HT) DNA genotyping technology. Leaf tissue samples were collected from varieties and advanced breeding lines that were used as parental material at the USDA- Agricultural Research Service’s sugarcane breeding programs at Houma, Louisiana (Ho) and Canal Point, Florida (CP), and the Louisiana State University Ag Center at St. Gabriel, Louisiana (L), as well as material currently in quarantine at Houma (Q).
The genotyping process involves a HT-extraction of leaf tissue DNA procedure. PCR amplification and capillary electrophoresis (CE) are processed semi-automatically using a liquid-handling Station, 384-well reaction plates, and fluorescence-labeled SSR primers. The CE files are analyzed using the GeneMapper™ software (Applied Biosystems, Inc., Foster City, CA). After excluding non-specific fluorescence peaks such as “stutters”, “pull-ups”, “dinosaur tails”, or “Minus-A”, a total of 144 robust, yet distinctive fluorescence peaks (SSR alleles) are chosen. These alleles are scored in a fixed sequential order, where presence of an SSR allele is scored as “A” and the absence of it as “C”. This sequential order from position 1 to position 144 of either “A” or “C” for any clone constitutes the molecular identity of that clone and is converted into a DNAMAN® (Lynnon Biosoft, Vaudreuil, Canada) file. Each file is named following the format of “Clone Name_LocationYear”. For example, “LCP85-384_Ho05” represents the molecular identity of LCP 85-384 from Houma in 2005, whereas “LCP 85-384_CP06” represented the molecular identity of LCP 85-384 from Canal Point in 2006. Upon constitution, the molecular identity was deposited into a sub-folder in the local database named after the year the sample was collected.
The molecular identity database is expandable to allow entry of newly assigned breeding lines each year. All or portions of the molecular identity files stored in the local database can be accessed and subjected to analysis by the Multiple Sequence Alignment Program of DNAMAN® (Lynnon Biosoft, Vaudreuil, Canada) to produce homology and/or phylogenetic trees with bootstrapping (confidence) values. When correctly labeled, different samples of any clone collected from different locations or different years join together as a group on the trees. Mislabeled ones are placed into different groups. In addition to enabling sugarcane breeders to identify mislabeled parental clones prior to their use as parents, the molecular identity database is being used to: provide molecular descriptors for newly released varieties, determine the genetic relatedness of elite clones, assess cross qualities, and identify F1 hybrids from (elite x wild) or (wild x elite) crosses.